J. Agric. Food Chem. 1996, 44, 613−616
613
Cotton Gossypium Spp. Plant Gossypol Contents of Selected GL2 and GL3 Alleles Jack C. McCarty, Jr., Paul A. Hedin,* and Robert D. Stipanovic Crop Science Research Lab, Agricultural Research Service, U.S. Department of Agriculture, Mississippi State, Mississippi 39762, and Southern Crops Research Lab, Agricultural Research Service, U.S. Department of Agriculture, College Station, Texas 77845
The gossypol content of cotton Gossypium spp. has been of interest to plant breeders because those that have high levels often carry resistance to the tobacco budworm Heliothis virescens (F.). However, its presence in seed has economic disadvantages to the seed and oil processing industry. Previously, two alleles which control plant gossypol, GL2 and GL3, were successfully transferred from several sources to a common background, XG-15. GL3 has now been transferred from G. raimondii (Ulbr.), a Peruvian wild diploid species, and G. davidsonii (Kell), a Mexican wild diploid species, into G. hirsutum (Upland) cottons. The plant bud tissue resulting from these crosses is relatively high in gossypol, while seed is relatively low. Keywords: Cotton; Gossypium spp.; plant gossypol; alleles INTRODUCTION
The gossypol content of cotton Gossypium spp. has been of interest to plant breeders because those that have high levels often carry resistance to the tobacco budworm Heliothis virescens (F.). However, its presence in seed has economic disadvantages to the seed and oil processing industry. It is of interest to determine whether the gossypol content of various plant tissues on which the insect feeds and develops could be increased without increasing or by even decreasing seed gossypol. The two main alleles that control the plant gossypol content are GL2 and GL3 (Bell and Stipanovic, 1977). Lee (1973, 1974) successfully transferred the GL2 and GL3 alleles from several sources to a common background XG-15. Lee et al. (1968) also reported that upland cotton plants monomeric for GL2 produced more gossypol in seeds than those monomeric for GL3; however, the disparity in seed gossypol production between cultivars of G. barbadense L. and the highest of the G. hirsutum L. stocks seems attributable to the greater expressivity of the GL3 allele in the upland cottons. Lukefahr and Martin (1966) and Wilson and Shaver (1973) reported that raising the terpenoid level in flower buds (squares) provided resistance to Heliothis spp. in upland cottons. It would appear that transfer of the GL2 and GL3 alleles from various available sources to upland cultivars could increase the gossypol content and provide insect resistance. This study is an investigation of the content of gossypol and other allelochemicals in leaves, squares, bracts, and seeds of 14 experimental lines, some for the first time, with one cultivar included as a baseline comparison. MATERIALS AND METHODS Cotton Sources. Cotton lines with various sources of the Gl2 and Gl3 alleles were tested. The Gl2 alleles were transferred from the following sources: (1) a high gossypol strain of G. hirsutum; (2) Coker 310, an upland cultivar adapted for
* Author to whom correspondence should be addressed [telephone (601) 323-2230; fax (601) 323-0915].
production in the South Atlantic states; and (3) H-1104, a “dooryard” G. hirsutum. The Gl3 allele was transferred from the following sources: (1) G. mustelinum Miers ex Watt, a Brazilian wild tetraploid; (2) G. tomentosum Nutt. ex Seem, a Hawaiian wild tetraploid; (3) Coker 310; 4) 3-T, an experimental strain created by M. J. Lukefahr (Lee, 1977) that has exceptionally high seed gossypol but relatively low flower bud gossypol; (5) G. raimondii Ulbr., a Peruvian wild diploid species; (6) G. davidsonii Kell., a Mexican wild diploid species; and (7) H-1014, a “dooryard” G. hirsutum from India. XG-15 is a high-gossypol experimental strain (Lukefahr and Houghtaling, 1969) of G. hirsutum that is the sibling of 3-T. This line harbors the Glr (rugate allele), a gene that conditions high boll glandulosity (Lee, 1978). Five to seven backcrosses were used to transfer Gl3 alleles to the XG-15 background. In addition to the above strains, monomeric (Gl2Gl2 gl3gl3 and gl2gl2 Gl3rGl3r), nullomeric (glandless, gl2gl2 gl3gl3), and dimeric XG-15 (Gl2Gl2 Gl3rGl3r) strains and Stoneville 213 (Gl2Gl2 Gl3Gl3), a commercial cultivar, were included in the test (Table 1). 1982-1994 Field Tests. The field tests were conducted in 1982, 1983, 1989, 1991, and 1994 on the Plant Science Farm at Mississippi State University. The cotton was planted each year about May 1 in single-row [0.97 × 12.8 m (W × L)] plots. Insects were controlled, as needed, with malathion and a synthetic pyrethroid. Analyses of Allelochemicals. Plant tissues (ca. 25 terminal leaves, squares, and bracts from each of two replications) were collected, freeze-dried, and ground. Allelochemical analyses were carried out following the procedures of Hedin et al. (1983a, 1991) for gossypol (phloroglucinol), flavonoids (diphenylboric acid-ethanalamine complex), and anthocyanins (absorbency at 540 nm of an aqueous alcoholic extract). Phloroglucinol was used for the analysis of gossypol as a substitute for aniline, which may be a health hazard. The results of the gossypol analyses were similar (Hedin et al., 1983b). The analyses were conducted in duplicate for each replication. Analyses were also performed using the anthrone procedure for total free sugars (Hedin et al., 1991). About 50 seeds were dehulled and ground prior to analysis for gossypol. Statistical Procedures. Data obtained from the various analyses and measurements were subjected to an analysis of variance, and lsd values were calculated according to SAS Institute (1985) methods. RESULTS AND DISCUSSION
In 1982 and 1983, preliminary field tests and analytical work were carried out on several lines in which the
This article not subject to U.S. Copyright. Published 1996 by the American Chemical Society
614 J. Agric. Food Chem., Vol. 44, No. 2, 1996
McCarty et al.
Table 1. Entry Number, Genotype, Source of Gl2 or Gl3 Alleles of Cotton Strains, and Abbreviations Used in Tables entry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
genotype gl2gl2 Gl3Gl3 gl2gl2 Gl3Gl3 gl2gl2 Gl3Gl3 gl2gl2 Gl3Gl3 gl2gl2 Gl3Gl3 gl2gl2 Gl3Gl3 gl2gl2 Gl3Gl3 Gl2Gl2 gl3gl3
source of Gl2 or Gl3 allele
abbrev
G. davidsonii G. tomentosum G. mustelinum G. hirsutum cv. Coker 310 G. hirsutum dooryard H1104 G. hirsutum strain 3-T G. raimondii G. hirsutum high-gossypol strain G. hirsutum cv. Coker 310 G. hirsutum dooryard H1014
Gl3 dav. Gl3 tom. Gl3 mus. Gl3 C310 Gl3 H1104 Gl3 3-T Gl3 rai. Gl2 h goss.
Gl2Gl2 gl3gl3 Gl2Gl2 gl3gl3 Gl2Gl2 gl3gl3 gl2gl2 Gl3rGl3r Gl2Gl2 Gl3rGl3r strain XG-15 gl2gl2 gl3gl3 Stoneville 213 glandless Gl2Gl2 Gl3Gl3 Stoneville 213
Gl2 C310 Gl2 H1014 2(Gl2gl3) 2(gl2Gl3r) XG-15 ST-213 gl ST-213
Table 2. Gossypol in Cotton Leaves, Squares, and Seed in 1982 and 1983 (Percent)a no.
entry
1 2 3 5 6 7 13 14 15
Gl3 dav. Gl3 tom. Gl3 mus. Gl3 H1104 Gl3 3-T Gl3 rai. XG-15 ST-213 gl ST-213
small leaves 1982 1983
flower buds 1982 1983
seed 1982
0.16 0.16 0.15 0.14 0.20 0.21
0.22 0.09 0.08 0.12 0.10 0.16
0.68 0.71 0.78 0.34 0.50 0.80
0.13 0.25
0.17 0.14 0.17 0.16 0.16 0.21 0.35 0.17 0.23
0.16 0.23
0.40 0.14 0.15 0.20 0.23 0.40 0.67 0.09 0.52
0.10
a A preliminary test with insufficient replication for statistical evaluation, percent of dry weight.
Gl2 and Gl3 alleles were transferred from various sources to a common background (Table 1). There was insufficient replication for statistical evaluation. The Gl3 allele from G. davidsonii and G. raimondii gave leaf and square gossypol levels comparable to those of the commercial cultivar Stoneville 213 (Table 2). The seed gossypol of the Gl3 entries ranged from 0.34 to 0.80%, whereas Lee (1977) had reported levels of 1-3% from several Gl2 and Gl3 lines. This difference should be attributed to the sources of plant material because results with the two gossypol reagents were similar (Hedin et al., 1983b). The Gl3 allele from G. davidsonii gave the best combination of square (0.22-0.40%) and seed (0.68%) gossypol. The other sources of the Gl3 allele did not seem to hold promise for increasing square gossypol while limiting the quantity in seed. Square gossypol was only slightly increased by the transfer of Gl2 sources. The leaf gossypol levels were only slightly higher than that of glandless levels in both 1982 and 1983. The square gossypol levels of the GL2 sources were about equal to the glandless levels in 1982 but were considerably higher in 1983. None of the GL2 sources appeared to be useful for increasing bud gossypol. The Gl3 allele from G. davidsonii and G. raimondii produced gossypol levels in flower buds that approached that of the cultivar ST-213. Tests on the effects of the Gl2 and Gl3 alleles on square and leaf gossypol were continued in 1989 and 1991. The results are summarized in Table 3. Analyses of the content of gossypol in seeds were performed in 1991 but not in 1989. Square gossypol was high both years in G. davidsonii and G. raimondii, sources of the GL3 allele. Both the monomeric gl2gl2 Gl3rGl3r and dimeric XG-15 resulted in increased square gossypol relative to ST-213. This is in agreement with previously published
Table 3. Total Gossypol in Cotton Plant Leaves, Squares, and Seeds, 1989 and 1991 (Percent)a 1991 1989 square leaf seed square gossypol gossypol gossypol gossypol
no.
entry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Gl3 dav. Gl3 tom. Gl3 mus. Gl3 C310 Gl3 H1104 Gl3 3-T Gl3 rai. Gl2 h goss. Gl2 C310 Gl2 H1014 2(Gl2gl3) 2(gl2Gl3r) XG-15 ST-213 gl ST-213
0.24 0.17 0.27 0.73 0.23 0.20 0.40 0.32 0.60
lsd 0.05 a
0.56 0.17
0.04 0.46
0.35 0.26 0.25 0.24 0.21 0.32 0.66 0.27 0.31 0.34 0.26 0.46 0.79 0.07 0.42
0.28 0.21 0.20 0.16 0.12 0.14 0.24 0.18 0.20 0.16 0.26 0.20 0.53 0.13 0.28
0.69 0.61 0.65 0.50 0.38 0.45 0.72 0.61 0.59 0.44 0.61 0.78 1.02 0.04 0.81
0.04
0.05
0.05
0.06
Phloroglucinol procedure.
data (Lee, 1977, 1978). Transfer of the Gl2 allele into the G. hirsutum lines did not result in appreciable increases in square gossypol, however. Leaf gossypol also was not appreciably increased upon transfer of the Gl2 allele to the G. hirsutum lines. In cotton plant tissues, gossypol is often the most prevalent of several terpenoid aldehydes (Stipanovic et al., 1984, 1988; Hedin et al., 1991). However, in seeds, gossypol is virtually the only terpenoid aldehyde present (Stipanovic et al., 1988; Khoshkhoo et al., 1994). For this work, it was considered of interest to compare the results of a reasonably specific colorimetric procedure, phloroglucinol (Hedin et al., 1991), to the HPLC procedure of Stipanovic et al. (1988) to determine whether recoveries from squares were similar and whether there was some unusual distribution of terpenoid aldehydes in any of the lines. Analysis of gossypol in squares of the 1994 lines by the phloroglucinol procedure gave somewhat lower, often variable, results that averaged 65% of the HPLC procedure (Table 4). Although the HPLC procedure is obviously more precise, the phloroglucinol procedure appears to be acceptable for preliminary screening because a plot of the phloroglucinol versus HPLC data was generally linear, particularly for the lower and higher analyses. These are the analyses of greatest interest. The ratios of the terpenoid aldehydes were fairly constant except for the lines having total terpenoid aldehyde (TTA) content quite different from the phloroglucinol value, which had higher heliocide or hemigossypolone (HGO) values compared with the GL3 dav. line. The TTA and phloroglucinol values for GL3 dav. and its HGO and heliocide values were fairly low. With the HPLC procedure, square gossypol accounted for 50-82% of the terpenoid aldehydes in the analyzed lines. Of the other components, hemigossypolone accounted for 3-8%, heliocide H1 6-17%, H2 4-13%, H3 2-5%, and H4 3-8%. From these data, it appears that the colorimetric analysis is adequate to evaluate the effects of the various alleles on the resulting gossypol content in squares. Tests to determine the effects of Gl2 and Gl3 alleles on plant tissue and seed gossypol contents were also carried out in 1994. In addition, analyses of two other insect allelochemicals, anthocyanins and flavonoids,
J. Agric. Food Chem., Vol. 44, No. 2, 1996 615
Gossypol Content of Cotton Species
Table 4. Total Gossypol in 1994 Cotton Squares As Analyzed by the Phloroglucinol Procedure and Total Terpenoid Aldehydes As Determined by Summation of the HPLC Maxima (Percent)a square terpenoid aldehydes, HPLC analysisb no.
entry
gossypolc
HGO
G
H1
H2
H3
H4
TTA
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Gl3 dav. Gl3 tom. Gl3 mus. Gl3 C310 Gl3 H1104 Gl3 3-T Gl3 rai. Gl2 h goss. Gl2 C310 Gl2 H1014 2(GL2gl3) 2(gl2Gl3r) XG-15 ST-213 gl ST-213
0.246 0.265 0.252 0.102 0.174 0.236 0.350 0.270 0.366 0.376 0.255 0.361 0.529 0.030 0.310
0.007 0.027 0.030 0.015 0.021 0.038 0.024 0.031 0.028 0.028 0.013 0.020 0.097 0.001
0.218 0.225 0.257 0.094 0.149 0.277 0.401 0.328 0.365 0.358 0.264 0.508 0.726 0.013
0.016 0.042 0.059 0.019 0.039 0.042 0.081 0.073 0.039 0.064 0.045 0.074 0.229 0.004
0.011 0.040 0.054 0.018 0.036 0.047 0.051 0.056 0.042 0.050 0.035 0.047 0.158 0.003
0.005 0.013 0.018 0.006 0.013 0.014 0.018 0.019 0.014 0.017 0.012 0.017 0.049 0.002
0.008 0.019 0.026 0.009 0.017 0.019 0.039 0.033 0.017 0.028 0.020 0.034 0.112 0.003
0.265 0.366 0.444 0.161 0.275 0.437 0.614 0.540 0.505 0.545 0.389 0.700 10.371 0.026
a Analysis expressed to three decimal places to accommodate HPLC data. b HGO, hemigossypolone; G, gossypol; H , H , H , and H , 1 2 3 4 heliocides, TTA, total terpenoid aldehydes. c Phloroglucinol procedure.
Table 5. Total Gossypol in Leaves, Squares, Bracts, and Seeds of 1994 Cottons (Percent) no.
entry
square gossypol
leaf gossypol
bract gossypol
seed gossypol
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Gl3 dav. Gl3 tom. Gl3 mus. Gl3 C310 Gl3 H1104 Gl3 3-T Gl3 rai. Gl2 h goss. Gl2 C310 Gl2 H1014 2(Gl2gl3) 2(gl2Gl3r) XG-15 ST-213 gl ST-213
0.20 0.22 0.20 0.18 0.19 0.22 0.32 0.23 0.32 0.33 0.27 0.37 0.56 0.05 0.33
0.13 0.13 0.14 0.12 0.12 0.14 0.21 0.13 0.14 0.17 0.17 0.14 0.27 0.06 0.16
0.05 0.05 0.06 0.04 0.07 0.05 0.09 0.08 0.06 0.07 0.07 0.07 0.16 0.06 0.08
0.55 0.52 0.46 0.77 0.60 0.59 0.59 0.84 0.95 0.14 0.70
lsd 0.05
0.046
0.023
0.017
0.03
0.71
a
Average of two sampling dates, Aug 4, 1994, and Aug 12, 1994, phloroglucinol procedure.
Selective combinations of gland-determining alleles can reduce (unfavorably) high amounts of toxic gossypol in cotton seed without reducing amounts in flower buds (Wilson and Shaver, 1973). These authors suggested that the genotype 2(gl2Gl3) be used, in which the source of Gl3 is G. raimondii. They had shown that this genotype reduced H. virescens larval growth without elevating seed gossypol levels. Our data would also suggest the efficacy of using Gl3 from both G. raimondii and G. davidsonii. These alleles gave flower bud gossypol levels that were equivalent to that of ST-213 but did not elevate seed gossypol levels. ACKNOWLEDGMENT
We thank Dr. J. A. Lee, retired, USDA-ARS, for furnishing seed of the different cotton stocks with the various combinations of Gl2Gl3 alleles, Lorraine Puckhaber for performing the HPLC analysis, and Moderia Petty for performing other analytical work. LITERATURE CITED
were carried out on plant tissue. The total sugar contents were also determined. The results of the analyses by the phloroglucinol procedure for total square gossypol in 1994 cottons (Table 5) were generally comparable to those performed in the previous years (Tables 2 and 3). The transfer of the Gl2 and Gl3 alleles to G. hirsutum lines did not result in higher gossypol. Total leaf, bract, and seed gossypol contents also were not markedly increased. Anthocyanins were analyzed because they had been shown to exist in part as a halo around gossypol glands (Hedin et al., 1983a) and had also been shown to be toxic to the tobacco budworm H. virescens (Fab.) in their own right. However, squares of all lines tested were low in anthocyanins (average 0.07%) except for XG-15 2(Gl2Gl3) (0.13%). Similarly, only this line possessed higher anthocyanin levels in leaves (0.55%; average 0.27%) and bracts (0.24%; average 0.14%). All lines were also analyzed for their flavonoid and sugar contents, but no helpful trends were identified. The average contents of flavonoids were as follows: squares, 0.37%; leaves, 1.04%; and bracts, 0.46%. The average contents of sugars were as follows: squares, 9.91%; leaves, 12.59%; and bracts, 11.89%. These data suggest that the alleles responsible for the increased gossypol content may not cause increases of other allelochemicals or metabolites.
Bell, A. A.; Stipanovic, R. D. The chemical composition, biological activity, and genetics of pigment glands in cotton. In Proceedings of the Beltwide Cotton Prod. Res. Conf., Atlanta, GA, Jan 10-12; National Cotton Council: Memphis, TN, 1977; pp 244-258. Hedin, P. A.; Jenkins, J. N.; Collum, D. H.; White, W. H.; Parrott, W. L.; MacGown, M. W. Cyanidin-3-β-glucoside, a newly recognized basis for resistance in cotton to the tobacco budworm. Experentia 1983a, 39, 799-801. Hedin, P. A.; Jenkins, J. N.; Collum, D. H.; White, W. H.; Parrott, W. L. Multiple factors in cotton contributing to resistance to the tobacco budworm Heliothis virescens (F.). In Plant Resistance to Insects; Hedin, P. A., Ed.; ACS Symposium Series 208; American Chemical Society: Washington, DC, 1983b, pp 347-365. Hedin, P. A.; Parrott, W. L.; Jenkins, J. N. Effects of cotton plant allelochemicals and nutrients on behavior and development of tobacco budworm. J. Chem. Ecol. 1991, 17, 11071121. Khoshkhoo, N.; Hedin, P. A.; McCarty, J. C., Jr. Terpenoid aldehydes in root-knot nematode susceptible and resistant cottonseeds as determined by HPLC and aniline methods. J. Agric. Food Chem. 1994, 42, 804-806. Lee, J. A. The inheritance of gossypol level in Gossypium II: inheritance of seed gossypol in two strains of cultivated Gossypium barbadense L. Genetics 1973, 75, 259-264. Lee, J. A. Some genetic relationships between seed and square gossypol in Gossypium hirsutum L. Proc. Beltwide Cotton Prod. Res. Conf. 1974, 87.
616 J. Agric. Food Chem., Vol. 44, No. 2, 1996 Lee, J. A. Inheritance of gossypol level in Gossypium. III. Genetic potentials of two strains of Gossypium hirsutum L. differing widely in seed gossypol level. Crop Sci. 1977, 17, 827-830. Lee, J. A. Allele determining rugate fruit surface in cotton. Crop Sci. 1978, 18, 251-254. Lee, J. A.; Cockerham, C. C.; Smith, F. H. The inheritance of gossypol level in Gossypium I: additive, dominance, epistatic, and material effects associated with seed gossypol in two varieties of Gossypium hirsutum L. Genetics 1968, 59, 285-298. Lukefahr, M. J.; Martin, D. F. Cotton pigments as a source of resistance to bollworm and tobacco budworm. J. Econ. Entomol. 1966, 59, 176-179. Lukefahr, M. J.; Houghtaling, J. E. Resistance of cotton strains with high gossypol content to Heliothis spp. J. Econ. Entomol. 1969, 62, 588-591. SAS Institute. SAS User’s Guide: Statistics, version 5 ed.; SAS Institute, Inc.: Cary, NC, 1985. Stipanovic, R. D.; Donovan, J. C.; Bell, A. A.; Matin, F. W. Factors interfering in gossypol analysis of okra and gland-
McCarty et al. less cottonseed using direct aniline extraction. J. Agric. Food Chem. 1984, 32, 809-810. Stipanovic, R. D.; Altman, D. W.; Begin, D. L.; Greenblatt, G. A.; Benedict, J. H. Terpenoid aldehydes in upland cottons: analysis by aniline and HPLC. J. Agric. Food Chem. 1988, 36, 509-515. Wilson, F. D.; Shaver, T. N. Glands, gossypol content, and tobacco budworm development in seedlings and floral parts of cotton. Crop Sci. 1973, 13, 107-110. Received for review August 31, 1995. Revised manuscript received November 20, 1995. Accepted December 11, 1995.X Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable. JF9505887
Abstract published in Advance ACS Abstracts, February 1, 1996. X
